The biosynthesis of the cross-linked peptidoglycan polymer in the inner cell wall of bacteria is a target of several classes of antibiotics. Structural modifications of peptidoglycan, such as alteration of the degree of cross-linking or amino acid substitution, have been observed in pathogenic bacteria that have developed antibiotic resistance. In a longterm collaboration, we have been using FAB-MS and CAD-MS/MS to characterize muropeptide monomers, dimers and trimers derived by enzymatic cleavage of the peptidoglycan. However, this technique has limited sensitivity for these molecules, particularly for higher oligomers. Moreover, MS/MS techniques often generate incomplete peptide sequence information and is not applicable to trimers and larger oligomers. We investigated the use of MALDI-MS and PSD analyses to overcome these limitations. However, conventional MALDI matrices are not successful in promoting ionization of muropeptides. A new class of matrices developed in our laboratory, based on mercaptobenzothiazole, have proven to be useful for MALDI-MS of muropeptides with much improved sensitivity relativae to FAB-MS. Even higher oligomers can be readily ionized and detected. Combining MALDI ionization with the new matrices and post-source decay (PSD) analysis has allowed peptide sequences and cross-linking patterns to be rapidly determined.